This competitive renewal grant is a request to support further analysis of age-related changes in membranes of human eye lens fiber-cells in order to elucidate major differences occurring in transparent and cataractous lenses. This proposal seeks to improve the methodology of membrane studies to the single lens level, which will allow us to consider donor health history information provided by the Eye Bank. The long-range goal is to understand the role of eye lens membranes in maintaining lens transparency. The lens membranes have unique lipid compositions and structures thought to maintain low oxygen concentration in the lens interior, and thus, protect against cataract formation. Our research will provide a basis to develop alternative strategies to prevent the onset or slow the progression of lens opacification. The emphasis will be on the role of the lipid bilayer portion of fiber-cell membranes in maintaining fiber-cell and lens homeostasis. Important progress in the previous grant period includes the identification of the role of cholesterol and the crucial role of cholesterol bilayer domains (CBDs) in particular. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis while the membrane phospholipid composition changes significantly with age. We will (i) continue to adapt, refine, and improve our recently developed methods for the quantification of lipid domains in intact fiber-cell membranes to membranes derived from a single lens, and (ii) based on single lens measurements, examine changes in fiber-cell membranes occurring with age and cataract formation. Special attention will be paid to determine major differences in the organization of lipids in lens membranes of people with cataracts and age-matched clear lenses, as well as to the structure of lens membranes of people who retain clear lenses into their eighth and ninth decades. Finally (iii), we will test the hypothesis that an increase in oxygen tension in the lens (one of the causes of cataract formation) initiates lipid peroxidation and drastically changes the organization of lipids in fiber cell membranes, including the formation of CBDs and cholesterol crystals. Our studies will be based on the use of state-of-the-art EPR techniques and methods available and developed at the National Biomedical EPR Center at the Medical College of Wisconsin. EPR spin-labeling methods permit identification of membrane domains, give information about structure and molecular dynamics as a function of the membrane depth in coexisting domains, and allow quantification of lipids in these domains. They also are capable of measuring oxygen transport within and across membrane domains.